tANCHOR-cell-based assay for monitoring of SARS-CoV-2 neutralizing antibodies rapidly adaptive to various receptor-binding domains

Summary Conventional neutralizing enzyme-linked immunosorbent assay (ELISA) systems for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mimic the protein-protein interaction between angiotensin-converting enzyme 2 (ACE2) and the receptor-binding domain (RBD). However, an easy and rapidly adaptative ELISA-based system for testing neutralizing antibodies against upcoming SARS-CoV-2 variants is urgently needed. In this study, we closed this gap by developing a tANCHOR-cell-based RBD neutralization assay that avoids time-consuming protein expression and purification followed by coating on ELISA plates. This cell-based assay can be rapidly adopted to monitor neutralizing antibodies (NAbs) against upcoming SARS-CoV-2 variants. We show that the results obtained with the tANCHOR-cell-based assay system strongly correlate with commercially available surrogate assays for testing NAbs. Moreover, this technique can directly measure binding between cell-surface-exposed RBDs and soluble ACE2. With this technique, the degree of antibody escape elicited by emerging SARS-CoV-2 variants in current vaccination regimens can be determined rapidly and reliably.


INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the cause of an ongoing worldwide pandemic that originated in Wuhan (China) and continued with the appearance of several viral variants, from alpha to omicron. 1,25][6][7] Until recently, all mRNA vaccine or vector regimens that efficiently reduce the risk of infection, severe disease, and death were based on the use of a spike sequence derived from the Wuhan and are adapted to the Omicron SARS-CoV-2 strain. 8,9The hallmark of RNA viruses is a high mutation rate that is beneficial for escaping neutralizing antibodies (NAbs).SARS-CoV-2 is an RNA virus in which mutations during replication and immune escape occur quickly. 10In particular, the receptor-binding domain (RBD) that interacts with the host protein angiotensin-converting enzyme 2 (ACE2) for cell invasion is constantly acquiring mutations. 11Fifteen of a total of thirty-seven mutations within the spike protein were identified within the Omicron B.1.1.529RBD. 12 Monitoring of immunoglobulin G (IgG) binding to the RBD with viral neutralization activity led to tests for antibody-mediated protection or vaccine efficiency.Upcoming SARS-CoV-2 variants can escape viral neutralization through mutation sites within the RBD. 13 Enzyme-linked immunosorbent assay (ELISA) systems based on the antibody-RBD antigen interaction are not sufficiently accurate to monitor SARS-CoV-2 neutralizing activity because, in such assays, all classes of antibodies, e.g., IgG with lower neutralizing activity than avidity-matured IgGs, are detected on the same target. 14Measurement of the protein-protein interaction (PPI) between ACE2 and RBD that is blocked by NAbs offers higher accuracy for measuring the neutralization activity and a higher correlation with the plaque reduction neutralization test (PRNT).6][17][18] However, an easy and rapidly adaptative ELISA-based system for testing NAbs against the RBD is needed.The global relevance of this topic has increased dramatically with the occurrence of upcoming variants of SARS-CoV-2.In the current study, we aimed to fill this gap by developing a cell-based neutralization assay that utilizes displayed SARS-CoV-2 RBD by the tANCHOR system and secreted soluble ACE2 (aa 1-740), including detection tags, to mimic the virus host PPI.The tANCHOR display system provides highly efficient and reliable presentation of heterologous proteins on the surface of human cells for antibody-binding studies and is based on the use of transmembrane domains (TMs) derived from the tetraspanin (Tspan) superfamily.Therefore, we choose this display system for anchoring the RBD on the cell surface in order to generate an antigen surface for testing of specific antibodies specifically directed against the RBD.A typical Tspan contains four TMs connected by intracellular, small, and large extracellular loops (ICL, SEL, and LEL). 19Importantly, the LEL is not necessary for routing a Tspan to the cell surface and can be replaced by heterologous protein sequences. 20,21In particular, the transmembrane anchors derived from the Tspan CD82 showed best Introduction of mutation sites within the RBD may influence the localization and expression level of tANCHORed RBDs.In order to identify such effects, we first coexpressed the ancestral Wuhan tANCHORed RBD fused with YFP together with the Wuhan, Alpha, Beta, Gamma, Delta, Lambda, and Omicron tANCHORed variant RBD fused with mCherry by transient transfection in HeLa cells.The colocalization of the tANCHORed Wuhan RBD fused to YFP was observed for all SARS-CoV-2 tANCHORed RBD variants fused to mCherry (Figures 1C  and S1).We used the fluorescence intensities of YFP and mCherry to calculate the Pearson correlation coefficient (PCC) for each pair.We observed no significant differences in the protein localization of the different tANCHORed RBDs (Figure 1D).Further, we quantified the expressed protein using the mCherry reporter (Figure 1E) and measured the emitted mean fluorescence intensity (MFI).We found no significant differences in the protein expression level between the variants compared with the Wuhan RBD (Figure 1F).Taken together, our data indicate that the RBD display using the tANCHOR system provides a robust and similar protein localization and expression level.

Stability of the cell monolayer during washing steps
Next, we tested if a cell monolayer could withstand washing steps using an automated ELISA washer because any ELISA system requires an automated ELISA washer when screening of large sample sets is desired.4][25] The cells were stained with crystal violet and followed-up the integrity of cell monolayers by microscopy and images of the 96-well plate bottom (Figure 2A).We observed that the staining and washing steps greatly affected the integrity of 293T monolayers.By contrast, HeLa cell monolayers were still intact even after five washing steps, and crystal violet in particular was washed out of the cells (Figure 2A).Even if HeLa cells are transfected with the plasmid ptANCHOR-CD82-Wuhan-mCherry, we have observed that the cell layers are still intact (Figure 2B).We intentionally did not use cell culture surface coatings such as poly-L-lysine for enhanced cell adherence because this step would make the assay laborious before cells could be seeded.The ability to use an ELISA washer with a low flow rate during cell-based ELISA techniques will enable the testing of large numbers of sera for neutralizing activity.

Secretion, characterization, and optimization of ACE2-V5-His binding conditions
We cloned the coding sequence of the human ACE2 protein for expression of the amino acids (aa) 1-740 fused to V5-6xHis tag in the expression vector FlexMam-Puro and named the vector pACE2 1-740 -V5-His (Figure 3A).This truncated version of the ACE2 ectodomain (aa 1-740) is known to be secreted into the cell culture medium. 26Secreted ACE2 1-740 -V5-His protein was detectable in the supernatant and in the cell lysates of transiently transfected HEK293T cells (Figure 3B, left).Generation of a HEK293T-based stable cell line producing ACE2 1-740 -V5-His showed stable and robust secretion of ACE2 1-740 -V5-His protein (Figure 3B left, top) that can be probed with antibodies against human ACE2 protein (Figure 3B, bottom).We further quantified the secreted ACE2 1-740 -V5-His protein from a pooled batch and obtained 7.0 mg/mL protein in the supernatant, which was isolated by a V5-nanoTrap, blotted to a membrane after staining with Coomassie dye, and probed with antibodies directed against the V5 epitope (Figure 3C).Next, we tested how much supernatant containing ACE2 1-740 -V5-His protein is necessary to obtain the highest possible absorbance value when binding was measured to the Wuhan RBD in our 96-well plate format.To this end, we transfected HeLa cells and tested different amounts of supernatant obtained from the stable cell line secreting ACE2 1-740 -V5-His protein.
We observed that binding to the expressed RBD on the cell surface is saturated at an applied volume of 100 mL to the wells, which corresponds to 0.7 mg ACE2 1-740 -V5-His protein (Figure 3D).A correction for background can be achieved by subtracting the absorbance obtained without adding ACE2 1-740 -V5-His from results where the receptor protein was added.The plasmid DNA amount that produced the highest absorbance value was also titrated by transient transfection with the plasmid ptANCHOR-CD82-Wuhan-mCherry.We found that a suitable DNA amount for transfection of HeLa cells with 1.0 mL of the transfection reagent are 300 ng per well of a 96-well plate (Figure 3E).Of note, cells were starting to detach when more than 300 ng/well was used for transfection.This is a sign of cell death that leads to lower amounts of ACE2 1-740 -V5-His binding.
We further optimized the binding assay of soluble ACE2 1-740 -V5-His to RBDs displayed on the surface of HEK293T cells by reducing background binding.We observed that applying a blocking solution containing 2% chicken egg albumin (CEA), 3% bovine serum albumin fraction V (BSA), and 10% normal goat serum (NGS) in 1X PBS (phosphate-buffered saline) produced the lowest background in the developed cellbased ELISA (Figure 3F).
A key application of our cell-based binding assays is neutralization ELISA in which the binding of ACE2 1-740 -V5-His to the RBD is blocked by antibodies in sera or plasma dilutions. 27,28The interaction platform with ACE2 is located within the RBD that is exposed on the SARS-CoV-2 full-length envelope spike protein.Our in vitro cell-based assay mimics the naturally occurring protein-protein interaction at the entry step of the SARS-CoV-2 spike protein with the host cell receptor ACE2. 29In order to evaluate how a difference in RBD expression levels influences the outcome of neutralization assays, we transfected HeLa cells with 0, 100, 150, 200, 250, and 300 ng of ptANCHOR-CD82-Wuhan-mCherry.Protein expression was assayed using the mCherry reporter and showed the expected correlation with the transfected amount of ptANCHOR-CD82-Wuhan-mCherry plasmid (Figure 3G).The obtained neutralization results at various tANCHORed RBD expression levels assayed with a convalescent serum as a source of NAbs were comparable (Figure 3H).This indicates that less difference in RBD expression levels does not significantly influence the neutralization result.
Next, we evaluate the serum dilutions of two convalescent individuals as well as a serum from a multiple vaccinated person and a prepandemic serum.As expected, the neutralizing activity decreases with increasing dilution and allows the determination of the dilution with 50% neutralization activity.(Figure 3I).Notably, a serum dilution of 1:10 produced an undesired background of measured neutralization activity when COVID-19 negative serum was used.An appropriate dilution to start with to test patient serum is therefore 1:20.

Binding of soluble ACE2 1-740 -V5-His protein on various SARS-CoV-2 RBD variants
The bound ACE2 1-740 -V5-His protein was visualized by indirect immunostaining on the cell surface of HeLa cells expressing the tANCHORed RBD of the Wuhan, Alpha, Beta, Gamma, Delta, Lambda, and Omicron variants.All displayed RBD were able to bind to the secreted ACE2 1-740 -V5-His protein (Figure 4A).Further, we tested differently transfected HeLa cells, which express one of the tANCHORed RBDs of Wuhan, Alpha, Beta, Gamma, Delta, Lambda, and Omicron SARS-CoV-2, respectively, in parallel assays for the binding efficiency of soluble ACE2 on the cell surface.This approach enables comparing the binding characteristics of ACE2 and the RBD variants in parallel.We observed a significant difference between the Wuhan and Delta and no significant differences between the Omicron and the Wuhan RBDs or between the Beta and Gamma RBDs.Additional significant differences were detected between the Alpha and Beta RBDs and between the Lambda and Omicron RBDs (Figure 4B).This comparison demonstrates that mutation sites within the RBD have an impact on the binding affinity of the ACE2 protein.For upcoming variants, this assay can therefore be used to characterize the interaction between the viral RBD and ACE2, which is useful to rate the infection profile of SARS-CoV-2.

Testing of sera from different groups by using the developed tANCHOR-cell-based ELISA for measuring neutralizing activity
For performing the optimized neutralization assay, HeLa cells are seeded in a 96-well format and transfected in order to display the RBD of choice on the cell surface, offering an interaction platform for ACE2.Incubation of serum from convalescent or vaccinated individuals will lead to binding of antibodies to the RBD, and free binding sites within the RBD that are not masked by NAbs are then detected by incubation with soluble ACE2 1-740 -V5-His protein.A low binding of ACE2 1-740 -V5-His will indicate the presence of NAbs.By contrast, highly efficient binding of ACE2 1-740 -V5-His will mean that antibodies are not able to block the interaction between the ACE2-and RBD-binding sites (Figure 5A).Using a panel of convalescent sera (n = 13), from individuals receiving one (n = 5), two (n = 10), or three (n = 10) doses of Pfizer-BioNTech BNT162b2 mRNA vaccine and COVID-19-negative sera (n = 8), we demonstrate that there are different potencies of neutralizing activity when different RBD variants are used for the neutralization assay (Figure 5B).We observed that the neutralization profile against all RBD variants, except the Omicron RBD, by antibodies derived from a three-vaccination schedule with BNT162b2 is comparable with the neutralization activity where the Wuhan RBD was used.Interestingly, all tested sera derived from convalescent individuals using the Wuhan RBD showed lower neutralization activity compared with the Alpha, Beta, Gamma, Delta, and Omicron RBD variants.An extremely low neutralization activity was observed overall when the Omicron RBD was used.The highest neutralizing activity was measured for individuals receiving three doses of BNT162b2 mRNA vaccine, with a mean value of 65.0% neutralizing activity for the Wuhan RBD.By contrast, the mean neutralizing activity value in the convalescent group was 62.8%.As expected, two doses of BNT162b2 resulted in the same neutralization profile for the convalescent group as observed by other reports. 30,31rrelation of the developed tANCHOR-based ELISA assay with a commercially available neutralizing ELISA system Next, we used the same serum panel to compare the developed SARS-CoV-2 neutralization assay with a surrogate ELISA assay similarly based on blockage of the PPI between ACE2 and the RBD, which is commercially available for testing NAbs (NeutraLISA, EUROIMMUN).This in vitro test produces results that are strongly correlated with plaque reduction neutralization tests (PRNT) using replication-competent SARS-CoV-2 particles. 32,33Unfortunately, this assay is only available for the Wuhan variant; therefore, we were only able to compare our neutralizing data for the Wuhan RBD.We performed the NeutraLISA assay as recommended by the manufacturer at a dilution of 1:5.However, testing the sera at a dilution of 1:20 allowed for better differentiation of the neutralization activity between the different groups (Figure 6A), and a high correlation (r = 0.89) between the tANCHOR-cell-based ELISA and the EUROIMMUN NeutraLISA assay was observed (Figure 6B).Further, we compared antibodies from convalescent individuals at an early stage (9-40 days after symptom onset) and a later stage (46-62 days after symptom onset) of infection and observed, for all RBD variants, higher neutralization activity at later infection stages (Figure 6C).We compared these two groups because IgG titers reach a plateau six weeks after symptom onset, and IgM titers decline between two and five weeks post-symptom onset. 34dditionally, ELISA performed against the SARS-CoV-2 nucleocapsid protein confirmed that only the convalescent group was literally infected with the pandemic SARS-CoV-2 virus (Figure 6D).In particular, testing antibodies directed against the nucleocapsid can differentiate between infected and vaccinated individuals. 35The sera obtained from vaccinated individuals in our sample subset therefore contained only antibodies raised against the RBD that were induced by the spike proteins expressed from the mRNA vaccine.

DISCUSSION
The initial outbreak of the SARS-CoV-2 pandemic has evolved into a world of variants where the ancestral SARS-CoV-2 strain, with its origin in Wuhan, China, has been outcompeted.This is the result of ongoing selection of SARS-CoV-2 variants that can replicate efficiently and evade the immune system.The target of most neutralizing antibodies (NAbs) produced by the immune system is the receptor-binding domain (RBD), which is essential for cell entry. 27,36,37This fatal infection step is critical for the virus; therefore, most selected mutation sites can be found within the RBD. 38Induced antibodies against the RBD bind to a specific epitope, and mutations within the epitope may enable the virus to escape the immune system.It is necessary to screen for NAbs against upcoming variants in order to surveil for SARS-CoV-2 variants that cannot be neutralized with antibodies induced by infection of a former circulating viral variant or, more importantly, vaccination regimes.The gold-standard technique for monitoring NAbs is the plaque reduction neutralization test (PRNT).However, this test requires the handling of an SARS-CoV-2 replication-competent virus in a BSL3 laboratory facility by well-trained staff and is time-consuming. 39,40seudotyped-virus-based assays require BSL-2 conditions, but this method is not suitable for high-throughput screening (HTS) and cannot be rapidly adapted to upcoming variants. 41,42Therefore, neutralization assays that are suitable for HTS without the use of infectious virus preparation are still needed.These are the reasons why ELISA-based assays mimicking the ACE-RBD interaction were developed.6][17][18] There are some commercially available assays that can measure NAbs in vitro.However, these assays cannot be rapidly adjusted to upcoming circulating variants because the development of a new ELISA system always requires the expression and purification of the RBD of the upcoming variant and the coating of the ELISA plate with this protein.This is obviously time-consuming, and companies are uneasy about investing financial resources in a new ELISA system because, in the coming months, an upcoming variant can potentially appear.A cell-based ELISA was developed, and the use of cells offering an ACE2 reaction surface for purified RBD protein was shown to be possible. 43owever, this assay cannot be rapidly adapted to the currently circulating SARS-CoV-2 variants because the RBD must be expressed and purified for a PPI test on Vero cells that express human-like ACE2.To avoid the need to express and purify a new RBD every time when a variant appears, we developed the tANCHORed-cell-based ELISA.The major advantage of this approach is that the transfected cells display the RBD on the surface, offering a PPI platform for ACE2 testing.Moreover, there is no need to purify the secreted ACE2 1-740 -V5-His from the supernatant.The generated stable cell line provides an inexpensive source of the soluble ACE2 interaction component for testing neutralizing antibodies directed against the RBD.Therefore, for all assay components, no additional isolation or purification step is necessary.Another advantage of our developed assay is that the interaction between ACE2 and the RBD can be directly compared.The only factor that influences this approach is the equal amount and quality of plasmid DNA.In line with other findings, we were able to confirm that the Omicron variant binds with equal affinity to ACE2 as reported for the Wuhan RBD; the highest binding affinity was observed on the Delta variant; and variants containing the mutation site N501Y in the Alpha, Beta, and Gamma RBD enhance ACE2-binding affinity (Figure 4B). 44,45ith none of the displayed RBD variants we did not observe any mislocalization or extreme differences in protein expression level (Figures 1E and 1F).For testing explicitly neutralizing activity in sera, our surrogate assay allows some degree of discrepancy in the displayed RBD protein amount (Figures 3G and 3H).This was expected because the reference for calculating neutralization activity is always the absorbance value at saturated binding of the displayed RBD variant by ACE2 1-740 -V5-His.This consideration is relevant because a different variant could possibly cause an altered expression of its tANCHORed RBD.In contrast, when ACE2 binding has to be measured and compared between RBD variants, the RBD-protein expression level must be additionally normalized.Although the transient transfection procedure is suitable for different variants where rapid results are needed, this developed assay should go a step further by generating stable cell lines expressing the indicated RBDs if time permits.Stable cell lines will certainly reduce the costs for performing the SARS-CoV-2 neutralization tests and will avoid transfection optimization.7][48] Our assay can support the PRNT in case of a prescreening method and more importantly, to compare neutralization of RBD-targeting and non-RBD-targeting neutralizing antibodies by using data from PRNT results.In addition, gain-of-function (GoF) research focused on the RBD variants that most efficiently escape the immune system in combination with increased ACE2 affinity can be performed without the risk of developing an infectious virus that can be potentially extremely pathogenic to humans.

Limitations of the study
There are certain limitations to the current study that require attention.One of the general limitations of the study that needs to be addressed is the principle of measuring the blockage of the interaction between the RBD and ACE2.Other possible neutralizing antibodies that are directed against the S2 domain or outside of the RBD region will not be detected by our developed cell-based neutralization assay.We cannot rule out the possibility that for upcoming SARS-CoV-2 variants, neutralizing antibodies outside the RBD region will become more important to estimate the neutralization activity.Additionally, we did not include any serum derived from SARS-CoV-2-asymptomatic individuals.So, the neutralization activity measured by the cell-based assay of those individuals is not known and should be addressed in future applications.
Secretion of ACE2 1-740 -V5-His protein HEK293T cells (6-well format) were transfected at a confluency of $80% with 4 mg plasmid DNA and 8 mL Metafectene (Biontex, Munich, Germany) according to the manufacturer's instructions.After 24 h, the medium was replaced by fresh medium containing 5 mg/mL puromycin (Carl Roth).Cells were treated after 3 days with a lower concentration of 2 mg/mL until enough viable cells were obtained from a 6-well plate.Cells were then detached with trypsin (Sigma Aldrich) and cultivated stepwise in larger flasks (25, 75, 150 mL) until enough cells were harvested for a 300 mL flask.Cells were harvested in a 300 mL flask until a confluency of 80% was reached, the medium was replaced, and 50 mL of medium was added.After 3 days, the supernatant was harvested, centrifuged to remove cell debris, pooled with another collected supernatant, and stored at À80 C until use.We used the same supernatant batch to perform all neutralizing assays.
Expression control and characterization of secreted ACE2 1-740 -V5-His protein by Western blot analysis One milliliter of supernatant from HEK293T cells transiently or stably transfected with the construct pACE2 1-740 -V5-His in a 6-well format was collected and centrifuged in order to separate cell debris from supernatants.Cleared supernatants were adjusted to pH 8.0 with 2 M NaOH solution (1-5 mL) and were added to 50 mL of a slurry of His tag isolation Dynabeads (Invitrogen) and incubated for 1 h at 4 C. Dynabeads were washed two times with wash buffer (1X PBS containing 0.05% Tween 20), and after magnetic separation, Dynabeads were eluted with 10 mL of elution buffer adjusted to pH 8.0, containing 300 mM imidazole (Carl Roth), 300 mM NaCl (Carl Roth), 50 mM Na 3 PO 4 (Sigma Aldrich), and 0.01% Tween 20.Eluted proteins were diluted with 20 mL of 2x Laemmli buffer.Cells were lysed in 100 mL 2x Laemmli buffer (Sigma Aldrich) containing 25 U of Benzonase (Millipore) and 1 mL HALT protease inhibitor cocktail (Thermo Fischer Scientific) in order to obtain cell lysates.After DNA degradation (3-5 min), His-tagged protein extracts were boiled for 3 min at 95 C and separated by SDS-PAGE using 4%-15% Protean TGX gels (Bio-Rad, Munich, Germany) and 1X SDS-PAGE buffer for electrophoresis.SDS-PAGE buffer (10x) contains 144.4 g Glycine (Carl Roth), 30.3 g Tris (Carl Roth), and 10 g SDS pellets (Carl Roth).Separated proteins were transferred to a 0.2-mm PVDF Mini trans-blot membrane (Bio-Rad) and then blocked for 30 min with 10% low-fat milk powder (Carl Roth) diluted in pure water.Proteins were detected using the specific primary antibodies 1:10,000 mouse anti-V5-HRP (1.18 mg/mL, Invitrogen), 1:10,000 anti-beta-actin-HRP (1 mg/mL, Thermo), or 1:5,000 rabbit anti-ACE2 (1 mg/mL, Invitrogen) with a 1-h incubation time.Secondary goat anti-rabbit-HRP (0.25 g/mL, Dako, Agilent Technologies, Denmark) was used at a dilution of 1:10,000 and incubated for 45 min.The activity of HRP was detected with Pierce West Pico substrate (Thermo Fisher scientific) and the Chemocam digital image analyzer (Intas, Go ¨ttingen, Germany).

Figure 1 .
Figure 1.Development of a cell-based ELISA specific for SARS-CoV-2 RBD neutralizing antibodies (A) Cartoon of the topology of the expressed tANCHORed SARS-CoV-2 RBD and expression construct design (not to scale).TM: transmembrane helices 1-4 derived from CD82, mCherry: C-terminal red fluorescence protein used as a reporter protein, RBD: receptor-binding domain, FLAG: N-terminal tag DYKDDDDK.DNA coding for the RBD SARS-CoV-2 variants was inserted into the tANCHOR vector using the EcoRI and EcoRV restriction sites.(B) Illustration of the mutation sites within the RBDs.(C) Representative confocal laser scanning microscopy (CLSM) images of HeLa cells 48 h post-transfection expressing the tANCHORed RBDs of Wuhan, Delta, and Omicron fused with YFP, or mCherry, which was used for the calculation of Pearson's correlation coefficient (PCC).Scale bars, 10 mm.See also Figure S1.(D) PCC calculation results (n = 10) showed no significant differences regarding protein localization between RBD variants and the ancestral Wuhan RBD.(E) Quantification of mCherry reporter protein (n = 4) indicates no significant effect of variant-specific RBD mutations on protein expression or (F) mCherry mean fluorescence intensity (MFI) (n = 6).(D-F) Significance was estimated by one-way ANOVA; non-significant (ns): >0.05; *p < 0.05, ****p < 0.0001.AU, arbitrary units.

Figure 2 .
Figure 2. Analysis of cell adherence during washing steps (A) Experimental steps for testing cell adherence during plate washing (left).Images of HEK293T or HeLa cell layers after staining with crystal violet and washing with an ELISA washer.(B) HeLa cells 48 h post-transfection with 0.3 mg plasmid DNA (ptANCHOR-CD82-Wuhan-mCherry) per well of a 96-well plate.Cell layers were fixed using 2% paraformaldehyde (PFA) as a fixative when tested for cell adherence during washing steps.Scale bars, 100 mm.

Figure 3 .
Figure 3. Characterization and binding analysis of soluble ACE2 1-740 -V5-His (A) Illustration of the ACE2 1-740 -V5-His expression construct (not to scale).ACE2: angiotensin-converting enzyme 2, V5-6xHis: C-terminal tag GKPIPNPLLGLDST fused with 6xhistidine.(B) ACE2 1-740 -V5-His transient protein expression in HEK293T cells (left) analyzed by western blot analysis using antibodies directed against the V5 epitope.Comparison of protein expression between transiently transfected HEK293T cells and the stable HEK293T cell line (right, top).Secreted ACE2 1-740 -V5-His protein from HEK293T stable cell line was probed with antibodies directed against human ACE2 protein (right, bottom).(C) Quantification of secreted ACE2 1-740 -V5-His protein obtained from a stably transfected HEK293T cell line.Bovine serum albumin (BSA) was used to generate a standard curve to identify the amount of secreted protein isolated by a V5 trap from the collected supernatant.Control contains supernatant from nontransfected HEK293T cells.(D) Determination of the supernatant amount for obtaining the highest absorbance value on the Wuhan RBD (HeLa cells transfected with 0.3 mg plasmid DNA per well).(E) Titration of the plasmid DNA amounts to reach the highest possible absorbance value for HeLa cells expressing the Wuhan RBD.(F) Testing of different blocking conditions using HeLa cells expressing the Wuhan RBD on the cell surface (HeLa cells transfected with 0.3 mg plasmid DNA per well).RNS, rabbit normal serum; NGS, normal goat serum; CEA, chicken egg albumin; BSA, bovine serum albumin; PBS, phosphate-buffered saline.(G) Results of cell-based neutralization assays (n = 4) using different plasmid DNA amount of ptANCHOR-CD82-Wuhan-mCherry.(H) Quantification of expressed mCherry fusion proteins (n = 4) from experiment (G).Expressed protein was collected for each quantification data point from three wells by cell lysis.(I) Impact on neutralization activity using different serum dilutions; each neutralization activity data point represents the mean of results from two wells.In each 96-well, 100 mL (0.7 mg protein) of ACE2 1-740 -V5-His-containing supernatant was used for performing experiments (E)-(G), and (I).Groups are compared for significance to the highest plasmid DNA amount used in the experiment by an unpaired two-tailed Student's t test; non-significant (ns): >0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5 .
Figure 5. tANCHOR-cell-based RBD ELISA for detecting neutralizing antibodies in different serological groups (A) The cartoon illustrates the workflow for the developed cell-based ELISA.(B) Results of using the developed cell-based ELISA to analyze human serum for neutralizing activity against the displayed Wuhan, Alpha, Beta, Gamma, Delta, Lambda, and Omicron RBD for SARS-CoV-2 RBD-specific NAbs.The neutralizing activity values were determined at 1:20 serum dilution.The dotted line represents the cutoff.The p value was calculated by an unpaired two-tailed Student's t test groupwise between Wuhan and other variants and within the Wuhan RBD; the significance is indicated on connection lines between the compared groups.non-significant (ns): >0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6 .
Figure 6.Validation of the developed cell-based ELISA (A) Sera was analyzed employing a commercially available neutralization assay (NeutraLISA assay from EUROIMMUN) at a dilution of 1:5 and 1:20.The range for negative specimens defined by the manufacturer is displayed as pale green background.(B) Comparison of neutralizing values obtained from NeutraLISA and tANCHOR-cell-based ELISA inhibition results in the full sample subset, Pearson's correlation coefficient (PCC).(C) Comparison of neutralization activity obtained for the Wuhan, Alpha, Beta, Gamma, Delta, Lambda, and Omicron RBDs between the early and late stages of SARS-CoV-2 infection.(D) Testing for antibodies against the nucleocapsid protein (NCP) for detection of SARS-CoV-2 infection in the used serum panel and two pre-pandemic controls.The dotted line represents the cutoff value; *p < 0.05.